Serological Survey Of Canine Influenza In Some Areas Of Guangdong And Virus Isolation And Identification

Canine influenza virus (CIV) is an emerging pathogen that causes acute respiratory disease in dogs. It has been reported that dogs can be infected by horse H3N8and avian H3N2influenza virus and the direct contact resulted in intraspecies transmission. Considering that dogs are in close contact with humans, whether the virus can spread from dogs to humans or not has caused considerable concerns for both veterinary and public health. Epidemiological investigation of canine influenza virus should be performed, which is important to the prevention and control of canine influenza.Serological test was performed on309dog serum samples of Guangdong province. Hemagglutination inhibition (HI) assay was used to detect the H3subtype antibody of CIV, and the total positive rate was26.21%. The seropositivity rates of pet dogs, farm dogs and stray dogs were34.02%,9.09%and25.93%, respectively. The exceptionally high seropositivity rate in some areas reflects the fact that H3N2canine influenza has become very popular in dog populations. This and other findings showed that the virus spreads rapidly and has an enlarging trend in recent years.29nasopharynx swabs from dogs with signs of respiratory disease were collected for virus isolation and identification. Two strains were isolated and designated as A/canine/Guangdong/12/2012(H3N2) and A/canine/Guangdong/23/2012(H3N2). Eight genes segments of them were amplified by RT-PCR method using specific primers. Then, the RT-PCR products were cloned into pMD19-T vector and sequenced. Phylogenetic analysis showed that eight gene segments of all the isolates had a close relationship with those of avian-origin H3N2canine influenza viruses reported from Thailand, South Korea and southern China. By comparing the deduced amino acid sequences of the HA and NA genes of the two isolates against the most similar avian strains, we found that the isolates had several common mutations at the receptor binding sites, potential glycosylation sites and cleavage site in HA, and antigenic sites in both the HA1and NA segments. An insertion of only one amino acid in the neuraminidase stalk was found in A/canine/Guangdong/12/2012(H3N2), which is different from other CIV isolates.In addition, the NP gene of H3N2CIV was cloned into vector pET-32a (+). The recombinant plasmid pET-NP was transformed into BL21(DE3) and induced to express with IPTG. Expression of nucleoprotein was examined and identified by SDS-PAGE and western-blot. The result s showed that the recombinant protein with the molecular weight of66ku was expressed successfully and they could react with positive serum against CIV particle. This provides a basis for the establishment of indirect ELISA method to test CIV antibody.In conclusion, this study applies a foundation for the further study on epidemiology, biological characterizations and detection methods of CIV.